Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, March 2001. / Includes bibliographical references. Also available on the Internet.

This thesis describes the development of biosensing systems incorporating eukaryotic cells. The ultimate objective of this work was to design devices capable of rapidly assessing the toxicity of effluents and environmental pollutants. Although much work remains to be done in order to achieve this goal, the work reported here demonstrates, in principle, the approaches adopted. The first approach exploited the reducing nature of healthy biological cells. So called 'redox mediated whole cell biosensors' have been described before. In this work, an algal toxicity test of short duration was developed and sensors incorporating cultured fish cells were described for the first time. The sensitivity of biosensors incorporating the green alga Selenastrum capricornutum, to diuron and pentachlorophenol, was found to compare favourably with that from other standard ecotoxicological tests. However, although the sensitivity of biosensors incorporating immobilised BF-2 fish cells was found to compare well with that of other fish cell-based toxicity tests, it appeared that whole organism tests were much more sensitive to the compounlds tested. The second approach involved the genetic manipulation of fish cells in order to incorporate luminescent reporter genes. Although this work is less well advanced, it demonstrates that the luc reporter gene can be successfully inserted into BF -2 fish cells and that these transformed cells can produce a luminescent response when incubated with luciferin substrate. Preliminary investigations have indicated that the sensitivity of luc-transformed BF-2 cells to 4-chlorophenol is comparable to that of some standard whole organism ecotoxicological tests and although much work is still required to validate this approach, it could eventually provide a simple, sensitive and rapid route to toxicity assessment.

The polyamines putrescine, spermidine and spermine play essential roles in the proliferation and differentiation of most eukaryotic cells. Inhibition of the polyamine pathway is known to have antitumour and antiparasitic effects and á-difluoromethylornithine (DFMO), a polyamine biosynthesis inhibitor, is clinically used in the treatment of African sleeping sickness caused by Trypanosoma brucei gambiense. Ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (AdoMetDC) are the rate-limiting enzymes in polyamine metabolism. Usually, these enzymes are individually regulated, however, in the malaria parasite, Plasmodium falciparum, these enzymes are part of a unique bifunctional PfAdoMetDC/ODC protein. In addition, compared to homologous proteins, this malarial protein contains six unique parasite-specific inserted regions, which can be targeted with novel drugs. A modified restriction enzyme-mediated inverse PCR method was developed to delete the largest parasite-specific insert (411 bp) from the large PfAdoMetDC/ODC gene (4257 bp). The method was compared to existing deletion mutagenesis PCR protocols and was shown to be the most effective method (80% mutagenesis efficiency) as opposed to the 40% positively mutated clones obtained with the overlapping primer method in deleting a >100 bp region. The independent removal of all three the PfAdoMetDC domain parasite-specific inserts and subsequent activity analysis thereof showed that these inserts are essential for the catalytic activities of both the decarboxylase domains. Plasmodia conserved secondary structures within these inserts were identified and were also shown to be very important for domain activities, possibly through protein-protein interactions across and within the domains of the bifunctional complex for the efficient regulation of intracellular polyamine levels. The N-terminally located O1 insert in the PfODC domain is a highly conserved and structurally distinct insert, which is essential for both domain activities. Previous studies showed that the deletion of this insert prevents dimerisation of the PfODC monomers and as a result influences association of PfODC with the PfAdoMetDC domain to form the bifunctional ~330 kDa complex. In addition, immobilisation of the insert via the mutagenesis of flanking Gly residues and the disruption of a single conserved α-helix within the insert severely affected both PfODC and PfAdoMetDC activities. It was thus hypothesised that the helix is involved in protein-protein interactions and the dimerisation of the PfODC domain. Size-exclusion chromatography of the monofunctional PfODC and bifunctional PfAdoMetDC/ODC proteins with disrupted helices resulted in the elution of only the monomeric (~85 kDa) and heterodimeric PfAdoMetDC/ODC (~160 kDa) proteins, respectively. The mono- and bifunctional wild type and immobile proteins eluted as both dimeric PfODC (~170 kDa) and heterotetrameric (~330 kDa) fractions as a result of intact protein-protein interactions. These results were subsequently exploited in the design and application of a parasite-specific, mechanistically novel, inhibitory peptide specific for this non-homologous insert in the bifunctional protein. A 1000x molar excess of a synthetic peptide, complementary to the α-helix within the O1 insert but opposite in charge, resulted in a ~40% inhibition of the PfODC enzyme. This study thus provides a proof-of-principle for the use of an inhibitory peptide targeting a parasite-specific insert in the dimerisation interface of a uniquely bifunctional malarial protein. / Dissertation (MSc)--University of Pretoria, 2008. / Biochemistry / unrestricted

Taking control of the protein production machinery of the host cell is a required step in the life cycle of viruses. Towards this end, viruses have evolved diverse strategies of cellular mimicry and deception to hijack and steal host cell ribosomes for viral protein production. In higher eukaryotes, where translation is sophisticated and access to ribosomes intricately regulated, numerous positive strand RNA viruses have evolved structured RNA sequences to evade translation regulation mechanisms. These RNA sequences, called Internal Ribosomal Entry Sites (IRESs), use their RNA structure to hijack the eukaryotic host cell ribosomes during the highly regulated initiation phase of translation. While a select few of such IRESs have been both biochemically and structurally characterized, the diversity of IRESs isn’t fully explored. Structural basis for the working mechanism of intergenic IRESs such as the Israeli Acute Paralysis Virus IRES (IAPV-IRES) with unique RNA features and expanded coding capacity is unavailable. Similarly, structural and biochemical understanding of newly described IRESs such as the complex IRES located at the 5′ untranslated region of the Cricket Paralysis Virus (CrPV 5′-UTR-IRES) is also unavailable. This body of work uses cryo-electron microscopy (cryo-EM) and biochemistry to characterize these two IRESs.Here, we show how the IAPV-IRES uses its unique features to exploit novel binding sites and commits the IRES-ribosome complexes towards a global pre-translocation mimicry. We trace a complete path of the IRES from its initial binding with the small subunit to its formation of an elongation-ready ribosome. We show that its mechanism of ribosome hijacking is different from currently accepted mechanistic paradigm for other IRESs from viruses similar to IAPV-IRES. We also identify another divergent mechanism of ribosome hijacking used by a different type of IRES. We show that the CrPV 5′-UTR-IRES features a novel, extended, and multi-domain architecture unlike any of the previously characterized IRESs from the group it belongs to. We also show that this IRES uses its novel structure and a minimal set of initiation factors to assemble a canonical-like pre-initiation complex on the small subunit of the ribosome at an upstream start-stop open reading frame. This body of work underscores the unexplored diversity in IRESs found in single stranded positive sense viral RNA genomes, invites re-visiting of the currently standing mechanisms of cap-independent initiation carried out by IRESs, and sheds light on a possible evolutionary past where IRESs could have given rise to the current eukaryotic translation initiation system.

In eukaryotic cells, DNA wraps around histones to form nucleosomes, which are the basic units of chromatin. Chromatin is classified as active euchromatin or repressive heterochromatin, depending on the modifications on histones and DNA. Heterochromatin, which is defined by the presence of histone modifications such as H3K9 methylation, serves important functions in cells such as silencing transposable elements, preventing aberrant recombination, and regulating gene expression.The fission yeast, which shares basic chromatin modification pathways with higher eukaryotes, is a premier model system for study heterochromatin formation. One important heterochromatin regulator is the JmjC-domain protein Epe1. It contains a conserved JmjC domain, which is commonly found in active demethylases. Despite that no in vitro demethylase activity has been demonstrated, Epe1 has been regarded as an H3K9 demethylase based on genetic evidence. However, the mechanism of its regulation is unclear at the beginning of my studies. In this thesis, I investigated the regulation of Epe1 through an unbiased genetic screen to identify factors important for Epe1 functions. From the screen, I identified multiple subunits within a transcriptional coactivator SAGA complex. I determined that Epe1 physically recruits SAGA to heterochromatin to promote histone acetylation and transcription, which provides a mechanism for a long-standing paradox regarding heterochromatin at repetitive DNA elements: heterochromatin normally represses transcription but the formation of heterochromatin requires transcription of the repeats. While past results suggest a role of Epe1 in promoting transcription of repeats, our results demonstrate how Epe1 promotes transcription. From this screen, I also identified multiple genes in the cAMP signaling pathway that are important for Epe1 function. I demonstrated that the cAMP signaling pathway regulates Epe1 protein levels post-transcriptionally, and this effect was also seen in cells experiencing glucose starvation, which dampens the cAMP signaling. This study uncovers another layer of control of Epe1 and provides a critical link between nutrient conditions and heterochromatin regulation. Altogether, my studies identified both a mechanism by which Epe1 promotes transcription within heterochromatin and a layer of Epe1 regulation by the glucose-sensing cAMP signaling pathway. These results will help future studies on Epe1 functions and how it is involved in epigenetic adaptation to changing nutrient conditions.

Synchronous cultures of Chlorella pyrenoidosa (strain 7- 11-05) have been utilized to measure the potential expression of the structural gene for isocitrate lyase (three D_S-Isocitrate glyoxylate-lyase, EC 4.1.3.1) during the cell cycle. Synthesis of the enzyme could be induced by placing cultures in the dark on acetate, with the induction process occurring in a quadratic fashion. By addition of cycloheximide during the course of induction, the increase in isocitrate lyase activity was shown to result from de novo protein synthesis. In the absence of protein synthesis the enzyme was stable for at least five hours. The pattern of uninduced isocitrate lyase synthesis during the cell cycle in continuous light, paralleled the stepwise increase of total cellular DNA. The enzyme appeared to be fully repressed for most of the cell cycle, and was derepressed during the time of DNA replication. Isocitrate lyase could be induced at all times in the cell cycle, indicating that the potential for gene expression is continuous in this eukaryote. A time lag was observed between the beginning of DNA replication and the initial rise in potential for isocitrate lyase gene expression. The control of gene expression in Chlorella appeared to be similar to that found in a fission yeast. / Ph. D.

Thesis (M.S.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains v, 61 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 50-56).

Mobile group II introns consist of a self-splicing RNA molecule and an intron-encoded protein with reverse transcriptase activity that function together in an RNP and catalyze the insertion of the intron into specific DNA target sites by a process known as retrohoming. The mechanism of insertion requires the intron RNA to bind and reverse splice into one strand of the DNA target site, while the intron-associated protein cleaves the opposite DNA strand and reverse transcribes the intron RNA. DNA target site recognition and binding are dependent upon base pairing between the intron RNA and the target DNA molecule. By modifying the recognition sequences in the intron RNA, group II introns can be engineered to insert into virtually any desired target DNA. Based on this technology, a novel class of commercially available group II intron-based gene targeting vectors, called targetrons, has been developed. Targetrons have been used successfully for gene targeting in a broad range of bacteria. Previously, our laboratory demonstrated that group II introns retain controllable retrohoming activity in mammalian cells, albeit with very low targeting efficiency. However, the gene targeting capability of group II introns is not limited to direct insertion of the intron. Group II introns can also create double-strand breaks that stimulate homologous recombination. By virtue of these attributes, mobile group II introns offer great promise for applications in genetic engineering, functional genomics and gene therapy. Here I present the results of experiments in which I tested group II introns for gene targeting activities in eukaryotic cells. First, I demonstrated that group II introns injected into zebrafish (Danio rerio) embryos retain in vivo plasmid targeting activity that is enhanced by the addition of magnesium chloride and deoxynucleotides. I also verified that similar in vivo targeting activity is retained in Drosophila melanogaster embryos. Further, I describe repeated experiments in zebrafish embryos designed to target the zebrafish genome with inconclusive results. Group II introns were also delivered to cultured human cells for genome targeting. Here I present promising evidence for the ability of group II introns to stimulate homologous recombination between an exogenously introduced donor DNA molecule and the chromosome. The donor DNA was delivered either as a linearized double-stranded plasmid by electroporation or as a single stranded genome of a recombinant adeno-associated virus (AAV). In both cases, cells receiving both the group II intron RNP and the donor DNA showed more efficient integration of the donor DNA than introduction of the donor DNA alone. The studies presented here provide insight into the potential of using group II introns for future applications in gene targeting in eukaryotes. / text

The molecules of life are small to us—billionths of our size. They move fast too, and in the cell they crowd together impossibly. Bringing that strange world into ours is the trick of molecular biology. One approach is to harness many copies of a molecule and iterate a reaction many times to glimpse what happens at that small, foreign scale. This is a powerful way to do things and has provided major insights. But ultimately, the fundamental unit of molecular biology is the individual molecule, the individual interaction, the individual reaction. Single-molecule bioscience is the study of these phenomena. Eukaryotic DNA replication is particularly interesting from the single-molecule perspective because the biological molecules responsible for executing the replication pathway interact so very intricately. This work is based on replication in budding yeast—a model eukaryote. The budding yeast genome harbors several hundred sequence-defined sites of replication initiation called origins. Origins are bound by the Origin Recognition Complex (ORC), which recruits the ring-shaped Mcm2-7 complex during the G1 phase of the cell cycle. A second Mcm2-7 is loaded adjacent to the first in a head-to-head orientation; this Mcm2-7 double hexamer encircles DNA and is generally termed the Pre-Replicative Complex, or Pre-RC. Mcm2-7 loading is strictly dependent on a cofactor, Cdc6, which is expressed in late G1. Much less is known about the details of downstream steps, but a large number of factors assemble to form active replisomes. Origin-specific budding yeast replication has recently been reconstituted in vitro, with cell cycle dependence mimicked by the serial addition of purified Pre-RC components and activating kinases. This work introduces the translation of the bulk biochemical replication assay into a single-molecule assay and describes the consequent insights into the dynamics of eukaryotic replication initiation. I have developed an optical microscopy-based assay to directly visualize DNA replication initiation in real time at the single-molecule level: from origin definition, through origin licensing, to replisome formation and progression. I show that ORC has an intrinsic capacity to locate and stably bind origin sequences within large tracts of non-origin DNA, and that ordered Pre-RC assembly is driven by Cdc6. I further show that the dynamics of the ORC-Cdc6 interaction dictate the specificity of Mcm2-7 loading, and that Mcm2-7 double hexamers form preferentially at a native origin sequence. This work uncovers key variables that control Pre-RC assembly, and how directed assembly ensures that the Pre-RC forms properly and selectively at origins. I then characterize replisome initiation and progression dynamics. I show that replication initiation is highly precise and limited to Mcm2-7 double hexamers. Sister replisomes fire bidirectionally and simultaneously, suggesting that previously unidentified quality control mechanisms ensure that a complete pair of replisomes is properly assembled prior to firing. I also find that single Mcm2-7 hexamers are sufficient to support processive replisome progression. Moreover, this work reveals that replisome progression is insensitive to DNA sequence composition at spatial and temporal scales relevant to the replication of an entire genome, indicating that separation of the DNA strands by the replicative helicase is not rate-limiting to replisome function. I subsequently applied this replication assay to the study replisome-replisome collisions, a fundamental step in the resolution of convergent replication forks. I find that, surprisingly, active replisomes absolutely lack an intrinsic capacity to displace inactive replisomes. This result eliminates the simplest hypothesized mechanism for how the cell resolves the presence of un-fired replisomes and has prompted and guided the development of alternate testable hypotheses. Taken together, these observations probe the molecular basis of eukaryotic inheritance in unprecedented detail and offer a platform for future work on the many dynamic aspects of replisome behavior.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, 2002. / Includes bibliographical references. Also available on the Internet.